Topically delivered 22 nt siRNAs enhance RNAi silencing of endogenous genes in two species.

MAIN CONCLUSION: 22 nt siRNAs applied to leaves induce production of transitive sRNAs for targeted genes and can enhance local silencing. Systemic silencing was only observed for a GFP transgene. RNA interference (RNAi) is a gene silencing mechanism important in regulating gene expression during plant development, response to the environment and defense. Better understanding of the molecular mechanisms of this pathway may lead to future strategies to improve crop traits of value. An abrasion method to deliver siRNAs into leaf cells of intact plants was used to investigate the activities of 21 and 22 nt siRNAs in silencing genes in Nicotiana benthamiana and Amaranthus cruentus. We confirmed that both 21 and 22 nt siRNAs were able to silence a green fluorescent protein (GFP) transgene in treated leaves of N. benthamiana, but systemic silencing of GFP occurred only when the guide strand contained 22 nt. Silencing in the treated leaves of N. benthamiana was demonstrated for three endogenous genes: magnesium cheletase subunit I (CHL-I), magnesium cheletase subunit H (CHL-H), and GENOMES UNCOUPLED4 (GUN4). However, systemic silencing of these endogenous genes was not observed. Very high levels of transitive siRNAs were produced for GFP in response to treatment with 22 nt siRNAs but only low levels were produced in response to a 21 nt siRNA. The endogenous genes tested also produced transitive siRNAs in response to 22 nt siRNAs. 22 nt siRNAs produced greater local silencing phenotypes than 21 nt siRNAs for three of the genes. These special properties of 22 nt siRNAs were also observed for the CHL-H gene in A. cruentus. These experiments suggest a functional role for transitive siRNAs in amplifying the RNAi response.

Systemic GFP silencing is associated with high transgene expression in Nicotiana benthamiana.

Gene silencing in plants using topical dsRNA is a new approach that has the potential to be a sustainable component of the agricultural production systems of the future. However, more research is needed to enable this technology as an economical and efficacious supplement to current crop protection practices. Systemic gene silencing is one key enabling aspect. The objective of this research was to better understand topically-induced, systemic transgene silencing in Nicotiana benthamiana. A previous report details sequencing of the integration site of the Green Fluorescent Protein (GFP) transgene in the well-known N. benthamiana GFP16C event. This investigation revealed an inadvertent co-integration of part of a bacterial transposase in this line. To determine the effect of this transgene configuration on systemic silencing, new GFP transgenic lines with or without the transposase sequences were produced. GFP expression levels in the 19 single-copy events and three hemizygous GFP16C lines produced for this study ranged from 50-72% of the homozygous GFP16C line. GFP expression was equivalent to GFP16C in a two-copy event. Local GFP silencing was observed in all transgenic and GFP16C hemizygous lines after topical application of carbon dot-based formulations containing a GFP targeting dsRNA. The GFP16C-like systemic silencing phenotype was only observed in the two-copy line. The partial transposase had no impact on transgene expression level, local GFP silencing, small RNA abundance and distribution, or systemic GFP silencing in the transgenic lines. We conclude that high transgene expression level is a key enabler of topically-induced, systemic transgene silencing in N. benthamiana.

Carbon Dots for Efficient Small Interfering RNA Delivery and Gene Silencing in Plants.

The initiation of RNA interference (RNAi) by topically applied small interfering RNA has potential applications for plant functional genomics, crop improvement and crop protection, but the primary obstacle for the development of this technology is the efficient delivery of RNAi effectors into the cell. The plant cell wall is a particularly challenging barrier for the delivery of macromolecules because many of the transfection agents that are commonly used with animal cells produce nanocomplexes that are significantly larger than the size exclusion limit of the cell wall. Here, we illustrate the use of a class of very small nanoparticles, called carbon dots, for delivering small interfering RNA into the model plants Nicotiana benthamiana and tomato (Solanum lycopersicum). Low-pressure spray application of these formulations with a spreading surfactant resulted in strong silencing of GFP transgenes in both species. The delivery efficacy of carbon dot formulations was also demonstrated by the silencing of endogenous genes that encode two subunits of magnesium chelatase, an enzyme necessary for chlorophyll synthesis. The strong visible phenotypes observed with the carbon dot-facilitated delivery were validated by measuring significant reductions in the target gene transcript and/or protein levels. Methods for the delivery of RNAi effectors into plants, such as the carbon dot formulations described here, could become valuable tools for gene silencing in plants with practical applications in plant functional genomics and agriculture.

[Groin pain in athletes]. / Liespijn bij sporters.

Groin pain in young athletes is a common problem, accounting for significant downtime in sports participation. It can be difficult to make the correct diagnosis as groin pain has a wide differential diagnosis, which encompasses acute as well as chronic causative factors. In this article this is illustrated by presenting three cases of patients who attended our hospital. In all three cases the main complaint was sports-related groin pain, and the patients presented with very similar symptoms. However, after further investigation the patients were diagnosed with three very different types of injury: sportsman's hernia; hip labral tear; and pubic osteitis. This emphasises the need for every general practitioner and medical specialist to understand that there is a wide differential diagnosis for groin pain in athletes, in order to be able to implement specific therapy targeting the actual cause of groin pain.

Excessive polyethylene wear and acetabular bone defects from standard use of a hooded acetabular insert in total hip arthroplasty.

PURPOSE: In total hip arthroplasty (THA) the use of a polyethylene (PE) insert with a hooded rim can be considered to reduce dislocation risks. This benefit has to be balanced against the potential introduction of impingement of the femoral component on this rim. We present a case series of early acetabular revisions for excessive PE wear and acetabular bone defects from overuse of such a hooded rim insert. MATERIAL AND METHODS: Twenty-eight patients with 34 consecutive early acetabular revisions were evaluated on failure mechanism. One type of implant was used in all cases. Standard pelvic radiographs and pre-operative CT scans were used to quantify PE wear, implant positioning and acetabular bone defects. RESULTS: An acetabular revision with impaction grafting was performed in all cases with a mean cup survival of ten years (range 1.3-19.3). No concurrent stem revisions were necessary. Overall implant positioning was adequate with a mean cup inclination of 45° (range 39-57) and anteversion of 25° (range eight to 45). The mean PE wear was 0.24 mm/year (range 0.00-1.17). The mean acetabular bone defect on pelvic CT scans was calculated as 352 mm² (range zero to 1107) and 369 mm² (range zero to 1300) in the coronal and transversal planes, respectively. A hooded acetabular insert was retrieved in all cases and profound PE wear, typically from the posterior hooded rim, was encountered. CONCLUSION: The use of hooded acetabular inserts may be considered to improve implant stability intra-operatively. This case series clearly presents that together with these devices, component impingement with concordant complications such as accelerated PE wear may be introduced. Standard use of these stabilizing inserts should thus be avoided.

The use of a dual-mobility concept in total hip arthroplasty patients with spastic disorders: no dislocations in a series of ten cases at midterm follow-up.

INTRODUCTION: Total hip arthroplasty (THA) is one of the treatment options in patients with cerebral palsy (CP) with painful osteoarthritis of the hip. However, the risk of dislocation of the prosthesis is higher in patients with CP when compared with physically normal patients. In this retrospective study of ten consecutive cases, we hypothesized that the use of a dual-mobility cup could reduce this risk of dislocation combined with good functional results. MATERIALS AND METHODS: From January 2008 until October 2010, eight patients (ten hips) with CP who consecutively received a THA using a dual-mobility cup were identified. At the time of surgery, the average age of the patient group was 54 years (range 43-61). Latest follow-up took place after on average 39 months (range 22-56 months). All patients or their caregivers were interviewed by telephone. They were asked if dislocation of the prosthesis had occurred. To evaluate quality of life and health in general, patients completed the SF-36 questionnaire. RESULTS: None of the prostheses had dislocated at the latest follow-up. Reoperation was needed in one patient after a periprosthetic fracture. Radiologic evaluation showed a mean cup inclination of 46 (range 27-58). On average, the quality of life of patients in this study was found to be limited in particular on the domains of physical health and functioning, while a fair to good score was measured at the six other different domains. CONCLUSION: The use of a dual-mobility cup in THA in patients with CP can lead to favourable results with respect to dislocation and clinical outcome.

Locking plate versus nonsurgical treatment for proximal humeral fractures: better midterm outcome with nonsurgical treatment.

BACKGROUND: Since its introduction, there has been controversy about the use of locking plates in the treatment of proximal humeral fractures. Have they really improved the functional outcome after a proximal humeral fracture or should nonsurgical treatment have a more prominent role? In order to evaluate our hypothesis that nonsurgical treatment for proximal humerus fractures should be the first choice of treatment, a matched controlled cohort study was conducted to compare the midterm (>1 year) functional and radiologic outcome of a group of patients treated with a locking plate and a matched group of patients treated nonsurgically. Complications in each group of patients were evaluated. MATERIALS AND METHODS: Through direct matching, 17 patients (1 bilateral fracture) treated with a locking plate were matched to 18 patients treated nonsurgically. Medical records and radiographs were reviewed retrospectively to obtain relevant patient related data and fracture type according to Neer classification (i.e. 2-, 3- and 4-part fractures). At the time of clinical follow-up, EQ-5D, American Shoulder and Elbow Surgeons (ASES) score, visual analog pain (VAS) pain and VAS satisfaction scores were completed. Active range of motion was tested. New radiographs were made to evaluate fracture healing, complications and, in the locking plate group, the position of the plate and screws. RESULTS: No significant differences were found in the characteristics of the patient groups. A significant difference in range of motion was found in favor of the nonsurgically treated patients. Results of ASES and patient satisfaction scores were also tending toward nonsurgical treatment. Furthermore, the complication rate was higher with locking plate treatment. Patients treated with a locking plate needed significantly more additional treatment on their injured shoulder (P = 0.005). DISCUSSION: This study's main limitation was the fact that the choice of initial fracture management was based on clinical judgement, as well as patient's fitness for surgery and therefore not randomized. By matching for fracture type this bias was largely overcome. Surgical treatment had a higher complication rate, requiring more additional treatment, which was often related to the initial surgery. Improving surgical technique could possibly lead to better outcomes for the surgically treated patients. In addition to the more favorable outcomes, nonsurgical treatment is also a more cost effective treatment. CONCLUSION: Nonsurgical treatment should have a more prominent role in the treatment of proximal humeral fractures.